S136: CONSTITUTIVE VLA-4 ACTIVATION IN CHRONIC LYMPHOCYTIC LEUKEMIA. THE OTHER SIDE OF BCR AUTONOMOUS SIGNALING.
Authors: E. Tissino, R. Bomben, P. C. Maity, F. Pozzo, G. Forestieri, A. Nicolò, A. Härzschel, T. Bittolo, F. M. Rossi, M. Datta, F. Zaja, G. Capasso, G. D’Arena, A. Chiarenza, F. Di Raimondo, H. Jumaa, D. Rossi, G. Del Poeta, T. N. Hartmann, A. Zucchetto, V. Gattei
Journal: HemaSphere (2022)

Background: In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator and key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. In CLL, beside the canonical antigen (Ag)-dependent mechanism, BCR signaling has been recently demonstrated to occur via an autonomous Ag-independent manner. Aims: To investigate the role of autonomous BCR signaling on constitutive VLA-4 activation state in CLL. Methods: VLA-4 activation/affinity was determined by flow cytometry (FC) using the conformation-sensitive anti-CD29 mAb HUTS21 and/or by “real-time” (FC) measuring the binding of the VLA-4 ligand LDV-FITC as reported (Tissino et al, J Exp Med, 2018) in: i) 1,984 consecutive CLL all with IGHV gene mutations/BCR features available; ii) sequential samples (0, 14, 30, 60 90 days) from CLL patients (n=26) treated in vivo with ibrutinib (IB) in real-world and from a clinical trial (NCT02827617). HUTS21 staining was also performed in the presence of: plasma depletion/replacement, soluble (s) VCAM-1, fibronectin (FN) and blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). BCR signaling were investigated by Ca++ influx assay in a 4-OH tamoxifen (4-OHT)-inducible murine TKO cell model (Dühren-von Minden M et al, Nature, 2012). Results: Out of 1,984 CLL, 1,070 (54%) expressed CD49d (cutoff 30%) and, among them, 250/1,070 (23%) were HUTS21+ (cutoff 20%), indicating an activated VLA-4 conformation. HUTS21 staining was: i) impaired by depletion of plasma from whole blood samples, and reconstituted by specific plasma components (sVCAM-1, FN); ii) impaired by pre-incubation with anti-CD49d HP1/2 blocking mAbs before addition of plasma, sVCAM-1 and FN. sVCAM-1 was higher in CD49d+ vs CD49d- CLL (p<0.0001); among CD49d+ cases, sVCAM-1 was lower in HUTS21+ (i.e. VLA-4 activated) cases (p=0.0096), suggesting ligand sequestration by activated surface VLA-4. CLL with mutated IGHV expressed higher levels of activated VLA-4 compared to unmutated IGHV CLL (p=0.001). Higher levels of activated VLA-4 were found in CLL using the IGHV3 and IGHV4 families, compared to cases using the IGHV1 family (p=0.043 and p=0.004). Finally, analysis of BCR stereotypy highlighted higher VLA-4 activation levels in CLL from subset#2 compared to CLL from subset#1 (p=0.02). To validate these data, murine TKO cells, expressing high VLA-4 levels, were transfected with different BCRs derived from 4 CLL with high level of constitutively activated VLA-4 (TKO-high) and 4 CLL with low level of constitutively activated VLA-4 (TKO-low). Compared to TKO-low cells, TKO-high cells showed a higher autonomous Ca++ influx (p=0.03), and consistently higher VLA-4 affinity (p=0.01). IB treatment impaired both BCR autonomous signaling and VLA-4 affinity. Notably, anti-IgM stimulation induced high Ca++ influx and high VLA-4 affinity state in both TKO-high and TKO-low, irrespective of IB treatment. According to TKO data, a decreased constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30, 60 and 90 from patients on IB, confirming an IB-dependent impairment of VLA-4 activation via BCR signal. Summary/Conclusion: The presence of a constitutively activated form of VLA-4 is observed in a fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from autonomous BCR signaling.